Stability and Fibrillation of Lysozyme in the Mixtures of Ionic Liquids with Varying Hydrophobicity.

Combinatorial effects of small molecules provide newer avenues to improve protein stability. The combined effect of two different classes of ILs on the stability and fibrillation propensity of lysozyme (Lyz) was investigated. Imidazolium-ILs (an aromatic moiety) with varying alkyl chains, methyl (MIC), butyl (BMIC) and hexyl (HMIC), and pyrrolidinium-IL (alicyclic moiety) with butyl substitution (BPyroBr) were chosen. The fibrillation was delayed by the addition of any of the IL. While added as a mixture with varying molar ratios, the presence of HMIC with MIC or BMIC at the ratio of 2:1 increased the fibrillation time synergistically by increasing lag time and reducing elongation rate. The protein stability was significantly reduced in these conditions compared to lower molar ratios of HMIC with MIC or BMIC. Molecular dynamics simulation studies indicated that upon adding Im-ILs water molecules were reduced around Lyz, whereas BPyroBr slightly increased the water around Lyz. Preferential interaction studies suggest that the preferential binding of HMIC with the protein was the most favored and it synergistically facilitated the preferential binding of MIC. Though BMIC was preferentially binding to the protein, it disfavoured the interaction of MIC. BMIC and BPyroBr had a competitive binding on the surface of Lyz. The results suggested that the mixture of ILs containing the longer alkyl chain destabilizes the protein and delays the fibril formation to a larger extent than the shorter alkyl chain ILs. Further, the effect of aromatic ILs could be greater than alicyclic ILs having the same alkyl chain length.